Mitochondria are the Achilles' heel of neurons. These long lived cells are especially dependent on mitochondria for energy production and metabolites, and dysfunction in this organelle is highly correlated with neurodegenerative disorders including Parkinson and Alzheimer diseases. Therefore, it is imperative that we understand how mitochondria are assembled from mainly nuclear-encoded components and how they normally function. Most mitochondrial proteins are imported from the cytosol and these pathways have been well characterized. In contrast, very little is known about RNA import despite the dependence of mitochondrial replication and function on small nuclear non-coding RNAs. Our group recently discovered that polynucleotide phosphorylase (PNPase) is a novel regulator of RNA import into mitochondria. Importantly, in a collaborative study we have recently linked a hypo-functional mutation in PNPase to a familial neurodegenerative disorder (von Ameln, et al, under review, Nature Genetics, 2011), strongly suggesting a specific requirement for RNA import in neurons. Thus, my overall scientific goal in this training proposal is to determine the function of PNPase in RNA import, to identify new components of the translocation pathway, particularly the mitochondrial outer and inner membranes channels, and to establish a role for PNPase-dependent RNA import in neural tissue. To achieve this goal, I will evaluate the function of PNPase mediated degradation in conferring import selectivity and assess whether known and putative stem-loop import sequences may protect imported RNA from PNPase-mediated degradation in Aim 1. The specific translocators of RNA inside the mitochondria will be identified by selectively blocking these factors through antibody treatments or using yeast mutants and performing RNA import assays in Aim 2. Finally, an inducible nestin-Cre recombinase x PNPase knockout mouse (PNP-nesKO) will be generated by crossbreeding, and the physiologic and pathologic roles of RNA import in neural tissue will be analyzed in Aim 3. Neurological, histological, and biochemical studies will be performed on these mice, with neuronal mitochondria analyzed for function. In addition to a human neurodegenerative disease linkage, this proposal is physiologically significant because deciphering RNA import requirements may lead to applications in treating mitochondrial diseases by targeted import of corrective RNAs. My studies aim to uncover a novel role for RNA import in maintaining neural tissue and its contribution to neurodegenerative diseases when this process is defective. PUBLIC HEALTH RELEVANCE: Defects in mitochondria have been implicated in a broad range of neurodegenerative diseases, emphasizing the importance of understanding fundamental aspects of mitochondrial assembly and function. Understanding and controlling RNA import provides an exciting new approach for treating mitochondrial diseases that are caused by mtDNA mutations because RNAs that substitute for the defective mtDNA could potentially be engineered for import into mitochondria.